LH-RH Activity Research Articles

Accuracy of serum luteinizing hormone and serum testosterone measurements to assess the efficacy of medical castration in prostate cancer patients

BackgroundLuteinizing hormone-releasing hormone (LH-RH) agonists are the standard for androgen deprivation therapy (ADT) in prostate cancer (PCa) patients. Current guidelines recommend serum testosterone measurement to assess the efficacy of ADT and to define castration resistance. However, serum testosterone does not reflect the exclusive effect of castration due to its extratesticular production. The aim of this study is to analyze if serum LH reflects better than serum testosterone the activity of LH-RH agonists.MethodsSerum LH and serum testosterone were measured with chemiluminescent immunoassay (CLIA) in a cohort study of 1091 participants: 488 PCa patients “on LH-RH agonists”, 303 “off LH-RH agonist” in whom LH-RH agonists were withdrawn, and 350 men with PCa suspicion “no LH-RH agonist” who never received LH-RH agonists. In a validation cohort of 147 PCa patients, 124 on “LH-RH agonists” and 19 “off LH-RH agonists”, serum testosterone was also measured with liquid chromatography and tandem mass spectrometry (LC MSMS).ResultsThe area under the curve (AUC) to distinguish patients “on versus off LH-RH agonists” was 0.997 for serum LH and 0.740 for serum testosterone, P < 0.001. The 97.5 percentile of serum LH in patients “on LH-RH agonists” was 0.97 U/L, been the most efficient threshold 1.1 U/L. The AUCs for serum LH, testosterone measured with CLIA and with LC MSMS, in the validation cohort, were respectively 1.000, 0.646 and 0.814, P < 0.001. The efficacy to distinguish patients “on versus off LH-RH agonists” was 98.6%, 78.3%, and 89.5% respectively, using 1.1 U/L as threshold for serum LH and 50 ng/dL for serum testosterone regardless the method.ConclusionsSerum LH is more accurate than serum testosterone regardless the method, to distinguish patients “on versus off LH-RH agonists”. The castrate level of serum LH is 1.1 U/l. These findings suggest that assessment of LH-RH agonist efficacy and castration resistance definition should be reviewed.

Ability of luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 binding to LHRH receptor on human liver cancer cells.

To explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) and LHRH binding to LHRH receptor (LHRHR) on the membrane surface of human liver cancer HEPG cells. LHRH was labeled by using (125)I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay. The specific activity of LHRH labeled with (125)I was 2.7 x 10(4) kBq/microL, and its radiochemical purity reached to 99.2-99.7%. The binding of (125)I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of (125)I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of (125)I-LHRH binding to the membrane LHRHR of HEPG cells, (125)I-LHRH of 1 x 10(5) cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40 and LHRH binding to the membrane LHRHR of HEPG cells were 0.43+/-0.12 nmol/L and 4.86+/-1.47 nmol/L, respectively, and their receptor volumes were 0.37+/-0.15 micromol/g and 0.42+/-0.13 micromol/g, respectively. The binding of LHRH-PE40 to the membrane protein of normal liver cells was not observed. The recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.

Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats.

The middle-age decline in reproductive function is manifested by reduced LHRH release, resulting in a decreased magnitude and delay of onset of the LH surge. Earlier studies suggested that the reductions in LHRH neural activation in middle-aged rats resulted from deficits in the afferent drive to the LHRH neurons. One critical afferent to the LHRH neurons lies in the anteroventral periventricular preoptic area (AVPv) nucleus. The neurons of the medial AVPv are synchronously activated to express Fos with LHRH neurons at the time of an LH surge in young adult animals. The present study examined whether, in middle age, reductions in the activation of AVPv neurons accompany the reduction in Fos activation in LHRH neurons. Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r(2) = 0.66; P < 0.01), suggesting that changes in the AVPv's drive to LHRH neurons underlie the decrease in LHRH activity in middle age. A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.

Changes in extracellular LHRH and beta-endorphin-like immunoreactivity in the nucleus infundibularis-median eminence of anestrous ewes under stress condition.

To clarify the effect of beta-endorphin released under stress condition on LHRH secretion, the concentration of LHRH and beta-endorphin-like-immunoreactivity (beta-END-LI) were analysed in perfusates from the nucleus infundibularis-median eminence of anestrous ewes subjected to footshocks stimulation. The dynamics of LHRH and beta-END-LI release during time-course of footshocks stimuli was altered. A brief facilitatory influence of stress on LHRH and beta-END-LI release was observed at the beginning of the stimulation on the first and third day; however the peaks of beta-END-LI were delayed about half an hour in relation to LHRH. Than on the first day of the stimulation LHRH returned to the control value but on the third day it declined below the control levels. Prolonged stress had stimulatory effect on beta-END-LI secretion during first and third day. The presented results indicate that: 1) short stress activates LHRH and beta-END-LI release, 2) prolonged stress has stimulatory effect on beta-END-LI release but leads to suppression of LHRH release. It is suggested that stress-induced beta-endorphin release may be one of the factors responsible for suppression of LHRH activity.

Structural requirements for α-mating factor activity

The sexual hormone of S. cerevisiae, α-mating factor (α-MF, WHWLQLKPGQPMY) has structural homology with mammalian luteinizing hormone releasing hormone (LHRH, pEHWSYGLRPG-NH 2) and has been shown to exhibit LHRH activity [Loumaye et al. (1982) Science 218, 1323–1325]. We have tested whether LHRH has α-MF activity in yeast and found that it does not. We therefore synthesized a series of hybrid peptides of α-MF and LHRH to study the structural features which determine α-MF and LHRH activities. A hybrid peptide consisting of the LHRH sequence with the C-terminal tetrapeptide (QPMY) of α-MF did not exhibit α-MF activity. Thus, the lack of α-MF activity of LHRH is not due solely to the absence of the C-terminal residues. Substitution of Lys 7 in α-MF with Arg, as is found in LHRH, did not affect the α-MF activity, nor did an additional substitution of Trp 1 with pGlu. However, the C-terminal four amino acids of α-MF were necessary for α-MF activity. Our results indicate that insertion of a Ser residue in position 4 as found in LHRH abolishes α-MF activity. These results suggest that, in addition to an intact C-terminus correct spacing of the N-terminal His 2 and the C-terminus is required for α-MF activity. The hybrid peptides all exhibited less LHRH activity than either LHRH or α-MF. These structure-function studies indicate that the structural homology between these two reproductive hormones may not reflect an evolutionary relationship between them.

Natriuretic peptides stimulate cyclic GMP production in an immortalized LHRH neuronal cell line.

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro. Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors--which represent GCs--we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.

Interleukin-1β inhibits the endogenous expression of the early gene c- fos located within the nucleus of LH-RH neurons and interferes with hypothalamic LH-RH release during proestrus in the rat

The ability of central interleukin-1β (IL-1β) administration to modulate the hypothalamic LH-RH release as well as the endogenous expression of the c- fos protein located within the nucleus of LH-RH neurons was examined during the afternoon of proestrus in rats. In a first series of experiments, 50 or 100 ng IL-1β were infused into the lateral ventricle of the rat brain at either 08.30, 12.00, 14.30, or 17.00 h of proestrus. The animals were then perfused transcardially with a solution of 4% paraformaldehyde from 17.30 and 18.00 h. In a second series of experiments, the rats were equipped with an intracerebroventricular (i.c.v.) cannula in the lateral ventricle and a push-pull cannula into the median eminence (ME), and LH-RH secretion was measured during the afternoon of proestrus. The third experiment investigated the putative role of corticotropin-releasing factor (CRF) in modulating the inhibitory effect of IL-1β on LH secretion by infusing CRF antagonists before the i.c.v administration of the cytokine to gonadectomized male and female rats. The central infusion of 50 or 100 ng IL-1β at 12.00 h completely blocked the spontaneous expression of c- fos protein which normally occurs in the nucleus of LH-RH neurons between 17.30 and 18.00 h on proestrus. In contrast, 50 ng IL-1β was less effective ( P < 0.05) when administered at 08.30 h, and totally without effect when infused at 14.30 h. Infusion of 50 ng IL-1β also markedly suppressed the hypothalamic release of LH-RH in proestrus rats bearing a push-pull cannula into the ME, and significantly decreased plasma LH levels in both gonadectomized male and female rats. Finally, we observed that the central administration of CRF antagonists did not modify the inhibitory effects of the cytokine on the activity of the hypothalamic-pituitary-gonadal (HPG) axis. These results provide the first direct evidence that IL-1β is a potent inhibitor of LH-RH neuronal activity during the proestrus LH surge in intact cycling rats. As central administration of this cytokine completely inhibited the endogenous expression of c- fos protein within the nucleus of LH-RH neurons, our findings also suggest that IL-1β acts at the level of LH-RH perikarya.

Sex steroids and the control of LHRH secretion

Gonadal steroids are important hormonal signals that regulate the activity of LHRH synthesizing and releasing neurons. Aside from a direct effect through the feedback mechanisms exerted at hypothalamic and/or anterior pituitary level, gonadal steroids may modify the rhythmic LHRH release by modulating other systems affecting LHRH neurons. 1. 1. In ovariectomized E 2-treated female rats, progesterone is able to evoke LHRH release from the perifused hypothalamus without affecting LH and FSH release. 2. 2. Excitatory amino acids (EAA) and their related analogs (NMDA and kainate) are known to stimulate LH release in young rats. When tested in a perifusion system on hypothalamic and anterior pituitary tissues, they differentially stimulate the release of LHRH (NMDA) and of LH (KA); their effect on both structures is markedly reduced following orchidectomy. It appears that gonadal steroids might exert a facilitatory action on the neurosecretory activity of LHRH neurons as well as a modulatory influence on the effect of EAA.

Acute Selective Withdrawal of Testosterone Negative Feedback Increases Luteinizing Hormone Secretion without Altering Hypothalamic Catecholaminergic euronal Activity*

Orchidectomy results in increased LH and FSH levels by removal of negative feedback at the hypothalamus and pituitary gland. However, the precise central nervous system mechanisms involved in elevation of gonadotropins after castration are unclear. We tested the hypothesis that catecholamine neuronal activity mediates the rise in serum LH that occurs after withdrawal of testosterone (T) negative feedback. The effects of acute and selective T withdrawal on brain catecholamine and LHRH activity and serum LH levels were determined in adult male rats. At the time of orchidectomy, rats were given sc implants of both T-containing and empty Silastic capsules. After recovery from surgery, the T-containing capsule was atraumatically removed from half of the animals (T-withdrawn), while the empty capsule was removed from the remaining rats (T-replaced). Rats were killed before and 6, 12, and 24 h after capsule removal. Serum T and LH levels were determined by RIA. Catecholamine content in microdissected nuclei of the LHRH neuronal system [medial preoptic nucleus, suprachiasmatic nucleus, retrochiasmatic area, arcuate nucleus (ARC), and median eminence (ME)] was measured by HPLC with electrochemical detection. Norepinephrine turnover rate was also determined in these areas by measuring the rate of decline of NE levels after blockade of synthesis with diethyldithiocarbamate. Additionally, LHRH content was measured by RIA within the ARC and ME. In T-replaced rats, the T capsules maintained serum T and LH levels within the normal range for intact male rats. In T-withdrawn rats, T levels fell into castrate range by 6 h after removal of the T capsule [0.12 +/- 0.04 ng/ml (mean +/- SEM); P less than 0.01 vs. T-replaced], and LH levels increased significantly from 0.23 +/- 0.04 ng/ml before capsule removal to 1.31 +/- 0.14 and 2.80 +/- 0.20 ng/ml 12 and 24 after T withdrawal, respectively (both P less than 0.01 vs. T-replaced). Despite a marked increase in serum LH levels, no significant changes in catecholamine content or NE turnover rate were observed in any of the hypothalamic nuclei of the LHRH neuronal system at any time after T withdrawal. ARC and ME LHRH levels also did not change significantly at any point after T withdrawal. These results suggest that activation of hypothalamic catecholamine neuronal activity is not required for the rise in serum LH levels after acute withdrawal of T negative feedback.

Correlation between changes in LH-RH content in the synaptosomal fraction of the olfactory bulb and some hypothalamic zones and level of sexual activity in male rats

We have studied the level of LH-RH in the synaptosomal fraction of the olfactory bulbs, preoptic area, and mediobasal hypothalamus, and the blood level of HL in adult male rats with sexual activity after noradrenergic denervation of the preoptic area using 6-oxydopamine. A neurotoxic effect of a 0.1% solution of ascorbic acid, preventing 6-oxydopamine disintegration was observed. Both sexual activity and level of LH-RH in synaptosomes of the preoptic area were reduced in male rats with noradrenergic denervation of the preoptic area. The maximum blood level of LH was observed during exposure to a recipient female rat, the LH-RH concentration in the synaptosomal fraction of the olfactory bulbs being raised, and that in the mediobasal hypothalamus being decreased. The results showed that LH-RH synthesizing cerebral neurons with terminals in the olfactory bulbs, preoptic area, and mediobasal hypothalamus are involved in the regulation of sexual behavior and the interaction of LH-RH-containing terminals with noradrenergic nerve endings at the level of the preoptic area.

Identification of the hypothalamic site through which locus coeruleus axons decussate to reach and stimulate contralateral LH-RH neurons

Recently we reported that locus coeruleus (LC) electrical stimulation (ES) amplifies the quantity of LH released prior medial preoptic nucleus (MPN)-electrochemical stimulation (ECS). In these studies we also observed that amplification of LH release occurred only when we activated those LC neurons whose cell bodies reside contralateral to the site of MPN-ECS. Seemingly, stimulatory LC axons decussate to reach contralateral hypothalamic regions which contain LH-RH neurons. The purpose of the present study was to identify the site(s) at which such decussation(s) occur. To accomplish this we used a special knife blade to make gross transections in hypothalamic regions previously described by others as regions where LC decussations occur. Transection 1 (TI) interrupted axons coursing through the medial forebrain bundle (MFB) in the region of the posterior lateral hypothalamus. This transsection was placed ipsilateral to the side of LC-ES and it had no effect on LH patterns of concentrations which were released by MPN-ECS. However, T1 completely blocked the amplifying effects of LC stimulation on LH secretion after MPN-ECS. Transection 2 (T2) was placed in the region of the MPN, parallel to the superior sagittal sinus. The knife blade was lowered in midline to the top of the 3rd ventricle and transected all fibers which cross midline within and around the anterior commissure. LH release following MPN-ECS was not appreciably affected in these rats nor did T2 alter the amplifying effects of LC-ES on LH. However, while plasma LH peaked between 60 and 75 min and then declined towards baseline in MPN + LC-stimulated rats, it remained significantly elevated throughout the remainder of the blood collection periods (180 min) in rats receiving combined MPN + LC and T2. Transection 3 (T3) also was placed in the MPN region and differed from T2 only in that we lowered the knife to the base of sphenoid bone. Thus, T3 disrupted all fibers which cross midline in the AC region and dorsal to the optic chiiasm (dorsal supraotic decussation of LC axons). This transection did not affect LH release evoked by MPN-ECS but completely eliminated the amplifying effects of LC stimulation after MPN-ECS on LH secretion. These data indicate that stimulatory LC axons which affect LH-RH neuronal activity enter the hypothalamus ipsilateral to the site of LC-ES and then project rostrally in the MFB to the lateral preoptic area. In this region, certain stimulatory LC fibers leave the MFB and cross midline in the dorsal supraoptic decussation to reach the contralateral MPN where they affect LH-RH neuronal activity. The data obtained in rats with AC transections (T2) also suggest that inhibitory inputs from extrahypothalamic regions enter the hypothalamus and suppress or arrest LH-RH release. Disruption of these inhibitory preoptic projections results in the sustained secretion of elevated levels of LH-RH/LH following MPN + LC stimulation.

Pulsatile Follicle-Stimulating Hormone Secretion Is Independent of Luteinizing Hormone-Releasing Hormone (LHRH): Pulsatile Replacement of LHRH Bioactivity in LHRH-Immunoneutralized Rats

We recently reported that passive immunoneutralization of endogenous LHRH in castrate male rats completely abolishes pulsatile LH secretion and, within 1 h, lowers mean plasma LH by 86%. While pulsatile FSH secretion, in terms of pulse amplitude and frequency, is not affected, mean plasma FSH is gradually lowered but only by 49% after 24 h. In the present study, we have examined the effect of replacing pulsatile LHRH biological activity on LH and FSH secretion in 4-week castrate male rats in which endogenous LHRH has been immunoneutralized by ovine anti-LHRH serum 772 (LHRH-AS) for 24 h. The LHRH-AS requires the 3-10 amino acid sequence of LHRH including the amidated C terminus for complete recognition. In order to circumvent the antiserum blockade, we utilized the LHRH agonist [Des Gly10]-LHRH ethyl amide (DG-LHRH) which is minimally recognized by the LHRH-AS but which possesses 2.6-fold the LH-releasing activity of LHRH. Twenty-four hours after injecting 500 microliter LHRH-AS into cannulated, castrate rats, sequential blood samples were taken every 10 min for 4 h. Bolus 3-ng injections of either DG-LHRH or saline were given iv either every 30 min during the 4-h collection period or every 30 or 60 min for 10 h before the initiation of and continuing through the 4-h collection period. Each DG-LHRH injection stimulated the release of a single pulse of LH, while pulsatile FSH secretion was unaffected. No synchrony was observed between the DG-LHRH pulses and the endogenous FSH pulses. Short term DG-LHRH treatment partially restored, and long term DG-LHRH treatment every 60 min completely restored, mean plasma FSH to the level observed in nonantiserum-treated castrate control rats. Long term DG-LHRH treatment every 30 min caused a rise in mean plasma FSH which exceeded the plasma FSH level of the nonantiserum-treated controls. The mean plasma level of LH was entirely dependent on the frequency of the DG-LHRH injection. The results of this study clearly demonstrate that pulsatile FSH secretion is independent of LHRH but that LHRH is required to elevate and/or maintain high mean plasma FSH levels. Trough levels of LH, however, are dependent on the frequency of LHRH-induced pulsatile LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrine studies in hyperprolactinaemic male mice.

In several species, including man and the rat, hyperprolactinaemia is associated with suppression of gonadotrophin release and male sexual behaviour. However, in the hyperprolactinaemic male mouse, plasma LH and FSH levels and copulatory behaviour are increased rather than suppressed. In an attempt to identify mechanism(s) which may be responsible for these effects of hyperprolactinaemia in the mouse, we have examined the effects of two ectopic pituitary isografts on several indices of hypothalamic and pituitary function in adult DBA/2J males. Animals with pituitary grafts had markedly increased plasma concentrations of prolactin, LH and FSH and enlarged seminal vesicles, whereas testicular and pituitary weights were not affected. Content of LHRH receptors and activity of aromatase in the pituitary, as well as dopamine-beta-hydroxylase activity in the hypothalamus were nearly identical in pituitary-grafted and sham-operated males. Biosynthesis of dopamine and turnover of noradrenaline in the median eminence were significantly increased in grafted males. We suggest that the increase in the activity of hypothalamic noradrenergic neurones may mediate stimulatory action of hyperprolactinaemia on LH and FSH release in the mouse. Comparison of these results with those obtained previously in the rat suggests that species differences in the effects of prolactin on gonadotrophin release may be related to its divergent effects on noradrenaline turnover.

Inhibitory effect of beta-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells.

To study the effect of human beta-endorphin (beta h-End) on pituitary response to gonadotropin-releasing hormone (LH-RH) and thyrotropin-releasing hormone (TRH) in vitro, we used dispersed rat pituitary cells. When beta h-End (10(-7) M) was simultaneously added along with LH-RH, its stimulatory effect was blocked and naloxone (NAL, 10(-5) M) did not reverse the beta h-End inhibitory effect. NAL alone elicited an increase in LH release, but in the presence of both stimulants (LH-RH and NAL), LH secretion was lower than that observed with LH-RH alone. TRH stimulatory activity of TSH and PRL secretion was blunted by the presence of beta h-End (10(-7) M) and was not reversed by NAL (10(-5) and 10(-3) M). These data suggest that beta h-End directly blocks the LH, TSH- and PRL-secreting activity of both LH-RH and TRH at the pituitary level. This beta h-End effect is not reversed by the specific opiate receptor blocker NAL.

Stimulation of hypothalamic LHRH levels and release by gonadal steroids

The inhibitory feedback effects of steroids on pituitary LH release are believed to be mediated via steroidal effects on the hypothalamic LHRH activity. We have examined the direct effects of individual steroids (T, DHT and E2) on hypothalamic LHRH levels and on LHRH release in vitro. In castrated male rats, replacement of either steroid in physiological doses, resulted in augmentation of the MBH LHRH levels by steroidal action within the MBH. LHRH analyses of microdissected diencephalic nuclei revealed that this accumulation occurred exclusively in LHRH terminals in the ME. Careful examination of the time course of steroid action showed that whereas LH release was suppressed within hours of steroid treatment, the LHRH response occurred after 3-4 days of steroid exposure in 2-week castrated rats and 7-14 days in 8-week castrated rats. This temporal dichotomy in the LH and LHRH responses to steroid action was further substantiated by the differential effects of low, sub-physiological levels of steroids on these two responses. Very low levels of T or E2 evoked maximal accumulation of the MBH LHRH, but LH release in vivo and the rate of LHRH release in vitro were not affected. Surprisingly, physiological levels of T which suppressed LH release concomitant with elevations in LHRH levels, augmented the in vitro rate of LHRH release. In fact, the LHRH release rate was found to be correlated with LHRH concentrations in hypothalami of intact, castrated and castrated rats treated with T. Thus it appears that in the hypothalamo-pituitary axis there are different thresholds of responsiveness to steroids. Apparently, the LHRH neurons, particularly the processes involved in LHRH accumulation are most sensitive to low levels of steroids; however, higher physiological levels of steroids are required to suppress pituitary LH release as well as to promote LHRH release. On the basis of our cumulative data, it is reasonable to speculate that steroid-induced accumulation of LHRH in the ME may not be a consequence of decrease in LHRH release, but may involve synthesis of the neurohormone.

Structure-activity relations of LHRH in birds.

Structural requirements in LHRH for gonadotropin-releasing activity were investigated by comparing the activity of the natural vertebrate LHRH structural variants and synthetic analogues. Substitution of Arg8 results in a loss of activity of LHRH in mammals, whilst a number of amino acid substitutions for Arg8 retain high gonadotropin-releasing activity in the chicken. Thus Arg8 of LHRH comprises an integral part of the binding site of LHRH and/or contributes towards the conformation of the binding site for the mammalian receptor while this does not pertain in the bird. The possibility that relative conformational stabilization of LHRH is important for biological activity in mammals but not birds, was supported by the demonstration that a gamma-lactam conformationally constrained analogue of LHRH was more active than LHRH in the mammalian system but equipotent in the bird. The chicken LHRH receptor is also relatively undiscriminating with regard to amino acid substitutions in positions 5 and 7. A series of LHRH analogues with pure antagonist activity in rats exhibited a spectrum of activities, from pure agonist to mixed activity and pure antagonist, in the chicken. These differences in LHRH structural requirements of the mammalian and avian receptor are reflected by a difference in molecular size of the chicken receptor (67,000) and mammalian receptor (60,000). Nevertheless, like the mammalian pituitary, the chicken pituitary does exhibit "desensitization" on prolonged exposure to LHRH.

Calcium is an inhibitor of luteinizing hormone-sensitive adenylate cyclase in the luteal cell.

Both prostaglandin F2 alpha (PGF2 alpha) and LHRH inhibit LH-stimulated cAMP accumulation and progesterone secretion in the intact luteal cell, but have no effect on LH-sensitive adenylate cyclase activity in isolated membranes. The present studies were conducted to assess the possibility that calcium (Ca2+) may mediate the inhibitory activity of PGF2 alpha and LHRH in the rat luteal cell. Removal of extracellular Ca2+ significantly enhanced cAMP accumulation in response to LH by about 2-fold, but blunted LH-stimulated progesterone secretion. Incubation of luteal cells with A23187 caused a highly significant and dose-related decrease in LH-stimulated cAMP accumulation with a concentration for half-maximal inhibition (IC50) of about 1 microM. No effect of A23187 was seen on LH-sensitive adenylate cyclase activity, but the ionophore elicited significant inhibition of LH-stimulated intracellular cAMP accumulation in the presence of isobutyl-methylxanthine (MIX), a phosphodiesterase inhibitor. Inhibition by A23187 was Ca2+ dependent, since a decrease in extracellular Ca2+ to less than 100 microM completely blocked the effect of the ionophore. A23187 also significantly inhibited LH-stimulated progesterone secretion in response to LH or cholera toxin and inhibited cholera toxin-stimulated cAMP accumulation in the absence or presence of MIX. In incubations of isolated luteal membranes, Ca2+ produced a dose-dependent inhibition of LH-stimulated adenylate cyclase activity in the absence or presence of MIX at free Ca2+ levels between 5-20 microM (IC50, approximately 10 microM). Depletion of extracellular Ca2+ had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha in the intact cell, and the inhibitory activity of LHRH was slightly reduced, but not abolished, by depletion of extracellular Ca2+. Verapamil, a Ca2+ channel blocker, had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha or LHRH. It is concluded that an acute increase in intracellular Ca2+ inhibits activation of adenylate cyclase by LH in the rat luteal cell. This conclusion is based on studies that showed enhanced cAMP accumulation by LH in Ca2+-depleted media, Ca2+-dependent inhibition of LH-stimulated cAMP production by a Ca2+ ionophore, and direct inhibition of LH-sensitive adenylate cyclase activity by Ca2+ in luteal membranes. It is suggested that a similar effect occurs in response to PGF2 alpha or LHRH in the luteal cell, but inhibition by these luteolytic agents is not dependent on an influx of extracellular Ca2+, but, rather, is due to an increase in intracellular Ca2+ by other mechanisms.

Effects of third ventricular injection of beta-endorphin on luteinizing hormone surges in female rat: sites and mechanisms of opioid actions in the brain.

The effects of third ventricular injection of beta-endorphin (beta-EP) on spontaneous, brain stimulation-induced and estrogen-induced LH surges were studied in the adult female rat. It was found that beta-EP blocked the preovulatory surge of LH release and ovulation, while it did not affect LH release in response to LH-RH injection. The site of the beta-EP blockade of ovulation was proved to be in the brain. Beta-EP completely blocked ovulatory LH release induced by the electrochemical stimulation of the medial amygdaloid nucleus and medial septum-diagonal band of Broca, but failed to block ovulation due to the stimulation of the medial preoptic area (MPO) or median eminence, though serum LH levels after the MPO stimulation were inhibited by beta-EP. In the spayed rats treated with estradiol benzoate (EB) on Day 1 and 4 of experiment, beta-EP given on Day 5 blocked the LH surge that normally occurred on that day and led to a compensatory surge of LH on the following day. Moreover, the LH surge on Day 5 was inhibited by beta-EP given either on Day 1 or Day 4. Present data suggest that beta-EP may act in inhibiting the preovulatory LH surges not only by suppressing the preoptic-tuberal LH-RH activities but also by affecting the initiation and development of stimulatory feedback of estrogen in the central nervous system.

Synthesis and biological activities of pseudopeptide analogues of LH-RH: Agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH 2S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the in vitro activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro 1, D-Phe 2, D-Trp 3,6]LH-RH antagonist was completely inhibitory at 30 ng in vitro and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed in vivo antiovulatory activity at the 250 μg level.

Comparative aspects of luteinizing hormone-releasing hormone structure and function in vertebrate phylogeny.

Immunoreactive LHRH was detected in hypothalamic and extrahypothalamic brain extracts of the rat, bird (pigeon and chicken), reptile (tortoise and lizard), amphibian (frog and toad), teleost (cichid), and elasmobranch (dogfish) and in the whole brain of the cyclostome (bagfish). The concentration of hypothalamic immunoreactive LHRH was more than 5-fold greater than that of the extrahypothalamic brain. Mammalian and amphibian hypothalamic immunoreactive LHRH yielded displacement curves parallel to those of synthetic LHRH in assays employing four antisera which recognize different regions of the decapeptide, thus suggesting a similarity in the structure of their LHRH. Hypothalamic immunoreactive LHRH from the bird, reptile, teleost, and elasmobranch differed from the mammalian and amphibian peptide in yielding displacement curves nonparallel to those of synthetic LHRH with three different antisera (1076, 743, and 744) which bind between Trp3 and Pro9 of LHRH. The differenece in structure appears to be near Leu7. With antiserum 422 which binds the NH2- and CO2H- termini of LHRH, bird, reptile, and teleost hypothalamic extracts yielded displacement curves parallel to that of synthetic LHRH. Bird, reptile, and teleost hypothalamic extracts showed displacement curves parallel to each other in all assays. In studies on the relative quantitation of LHRH, all four antisera gave similar values of immunoreactive LHRH concentration in mammalian hypothalamic extracts and in amphibian hypothalamic extracts. By contrast, assay of bird, reptile, teleost, and elasmobranch hypothalamic extracts with antiserum 422 gave much higher values than did the other antisera, suggesting that the LHRH peptide is structurally different from mammalian and amphibian LHRH in the region of Leu7 but similar at the NH2- and CO2H-termini. These conclusions are supported by studies on the biological activity of hypothalamic LHRH from the different species using dispersed ovine anterior pituitary cells in culture. The LH release responses to equivalent amounts of immunoreactive LHRH (as measured by antiserum 422 which binds a region of LHRH essential for biological activity) from the various species were similar, indicating that the biologically active region of the molecule has been conserved in evolution. Structural differences in vertebrate hypothalamic immunoreactive LHRH were confirmed by cation exchange and high pressure liquid chromatography. Our findings of differences and similarities in vertebrate LHRH support a contemporary phylogenetic scheme.